Heart disease is the primary cause of death in most western societies. The most prevalent heart disease states are related to platelet-dependent ischemic syndromes, including, but not limited to atherosclerosis and arteriosclerosis, acute myocardial infarction, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic cardiovascular devices (e.g., in-dwelling catheters or shunts "extracorporeal circulating devices"). These syndromes represent a variety of stenotic and occlusive vascular disorders thought to be initiated by platelet activation either on vessel walls or within the lumen by blood-borne mediators.
Circulating platelets have been shown to play a central role in the response to a variety of blood vessel injuries, such as narrowing of the lumen, plaque formation, and the presence of foreign bodies (e.g., catheters) and the like. The response of platelets to these injuries is a sequence of events including platelet adherence, platelet aggregation, formation of microthrombi, and release of platelet granular components, including potent cellular mitogenic factors. There is a variety of pathologies that can occur from this sequence of events, including a variety of atherosclerotic and thrombotic phenomena.
Integral to platelet function is the platelet membrane glycoprotein (GP) IIb-IIIa complex which constitutes the fibrinogen (Fg), von Willebrand Factor (vWF), and fibronectin (Fn) receptor on activated platelets (see Phillips et al., Blood (1988) 71:831-843). Platelet GP IIb-IIIa is now known to be a member of a superfamily of structurally and functionally related adhesive protein receptors known collectively as the "integrins". Many stimuli have been identified which are thought to activate the GP IIb-IIIa receptor on the platelet surface and thus lead to the aggregation of platelets in vivo, and ultimately to the formation of thrombi. These stimuli include ADP, epinephrine, thrombin, collagen and thromboxane A.sub.2. The activated GP IIb-IIIa complex on stimulated platelets binds the adhesive proteins Fg, Fn and vWF; however, it is the binding of fibrinogen that is believed to be principally responsible for platelet aggregation and thrombus formation in vivo. Therefore, substances which specifically inhibit the binding of fibrinogen to GP IIb-IIIa inhibit platelet aggregation and could be candidates for inhibiting thrombus formation in vivo.
Short peptides derived from the sequences of Fg, Fn and vWF have been disclosed which block the binding of these adhesive proteins to activated platelets and inhibit platelet aggregation (see Hawiger et al., U.S. Pat. Nos. 4,661,471; and Rouslahti et al., U.S. Pat. No. 4,614,517). One of these peptides is the sequence RGD, and the tetrapeptide sequence RGDS has been used specifically. The amino acid sequence RGDX is found in a variety of adhesive proteins and has been demonstrated to play an important role in the interaction with adhesive protein receptors. See, e.g., Pierschbacher et al., J Biol Chem (1987) 262:17294-17298; Ruggeri et al., Proc Natl Acad Sci (USA) (1986) 83:5708-5712; and Ruoslahti et al., Cell (1986) 44:517-518. A separate class of inhibitory peptides utilizes peptide sequences modeled on the carboxyl terminal sequence derived from the gamma chain of fibrinogen, the dodecapeptide HHLGGAQKAGDV (Kloczewiak et al., Biochemistry (1989) 28:2915-2919; Timmons et al., Ibid., 2919-2923). However, the usefulness of the RGD and dodecapeptide-based small peptides is limited because they either have a low affinity of interaction with platelet receptors (IC.sub.50 =10-100 uM) or interact with other adhesive protein receptors.
Recently, several groups have isolated and characterized low molecular weight polypeptide factors from snake venoms which have extremely high affinity for the GP IIb-IIIa complex on stimulated platelets. Huang, T.F., et al., J Biol Chem (1987) 262:16157-16163; Huang, T.F., et al., Biochemistry (1989) 28:661-666 report the fibrinogen and/or von Willebrand Factor platelet binding inhibition properties and primary structure of trigramin, a 72 amino acid peptide containing RGD and 6 disulfide bridges isolated from Trimeresurus gramineus. Gan, Z.-R., et al., J Biol Chem (1988) 263:19827-19832, report the properties and structure of echistatin, a 49 amino acid peptide also containing RGD and 4 putative disulfide bridges which is isolated from Echis carinatus. Williams, J. A., et al., FASEB Journal (1989) 3:A310, Abstr. No. 487m, report the related peptides elegantin, albolabrin, and flavoviridin. All factors thus far purified from snake venom which inhibit the binding of adhesive proteins to integrin receptors contain the RGD sequence.
Although these reported snake venom factors are potent platelet aggregation inhibitors in vitro, these peptides also bind with high affinity to other members of the adhesive protein receptors such as the vitronectin and fibronectin receptors (Knudsen, K. A., et al., (1988) Exp Cell Res 179:42-49). This lack of specificity of snake venom factors thus far identified for GP IIb-IIIa is an undesirable feature of their therapeutic use as inhibitors of thrombus formation.
Another approach for the generation of thrombus inhibitors has been the development of murine anti-GP IIb-IIIa receptor monoclonal antibodies which block the binding of the adhesive proteins to stimulated platelets. Use of these monoclonal antibodies to prevent coronary artery reocclusion after reperfusion with tissue plasminogen activator in dogs has been reported (Yasuda, T., et al., J Clin Invest (1988) 81:1284-1291).
Clearly, additional therapeutic treatment regimens are needed for preventing or at least mitigating undesirable thrombus formation. In particular, therapeutic agents capable of blocking or inhibiting thrombus formation at specific locations would provide major therapeutic benefits. Ideally, these agents will be potent, specific and nonimmunogenic to most patients; they should be easy to administer, stable and economical to produce. Further, these agents should act transiently and be capable of functioning at the earliest stages of thrombus formation, without interfering with long-term hemostasis. The present invention fills these and other related needs.